The present invention relates to a method for induction of differentiation of monocytes to osteoclasts, an agent comprising monoclonal antibody used for the above method, and a method for screening a ligand that induces differentiation of monocytes to osteoclasts.
It is known that osteoclasts are multinucleated giant cells involved in bone resorption and have their origin in hematopoietic system (Udagawa, N. et al., Proc. Natl. Acad. Sci. USA 87: 7260-7264 (1990)). Although precise mechanisms for their differentiation and activation are not clear at present, osteoclasts are one of the most important factors that participate in the physiological metabolism of the bone and abnormality of these cells is related with osteoporosis. Formation of multinucleated cells induced by membrane fusion is the first step of formation of osteoclasts. Membrane fusion is an important process in biological phenomena (White, J. M., Annu. Rev. Physiol. 52: 675-697 (1990), Burger, K. N. and Verkleij, A., J. Experientia 46: 631-644 (1990)). Membrane fusion is also involved in fertilization, formation of muscles, exocytosis, endocytosis, formation of organella, and transport between intracellular organella. However, mechanisms that control membrane fusion is not well understood yet.
It has been reported that interstitial cells and steroids such as 1a, 25-dihydroxyvitamin D3 or parathyroid hormone are necessary for formation of multinucleated osteoclasts caused by fusion of mononuclear cells of macrophages/monocytes (Takahashi, N. et al., Endocrinol. 122: 1373-1382 (1988), Takahashi, N. et al., Endocrinol. 123: 1504-1510 (1988), Quinn, J. M. W. et al., The Endocrin. Soc. 134: 2416-2423 (1991)). It has also been reported that several kinds of cytokines induce the formation of multinucleated cells from monocytes/macrophages (Lacey D. L. et al., Endocrinol. 136: 2367-2376 (1995), Murray G. R. J., Bone and Miner. Res. 8: S505-S510 (1993)). However, these methods do not clearly demonstrate that derived cells perform bone resorption. Furthermore, these methods require the presence of stroma cells and, thus, are not simple. There has been no report pertaining to a method of in vitro induction and differentiation of osteoclasts.
An object of the present invention is to provide a simple method for production of osteoclasts from monocytes, a agent comprising monoclonal antibody used in this method, and a method for screening of a ligand that induces differentiation of monocytes to osteoclasts. To achieve the object, the present inventors ardently studied and succeeded for the first time in differentiation of monocytes to osteoclasts in vitro by culturing isolated monocytes in the presence of monoclonal antibodies that bind to FRP-1 protein present on the surface of monocytes. Furthermore, the present inventors found that it was possible to isolate a ligand that binds to FRP-1 protein and induces differentiation of monocytes to osteoclasts with the use of the system for differentiation of monocytes to osteoclasts.
Thus, the present invention relates to a simple method for induction of differentiation of monocytes to osteoclasts using monoclonal antibody capable of inducing differentiation of monocytes to osteoclasts, an agent comprising the monoclonal antibody, and a method for screening a ligand that induces differentiation of monocytes to osteoclasts. More specifically, the present invention relates to
(1) a method of producing osteoclasts which comprises a step of contacting monocytes with monoclonal antibody that binds to a portion of FRP-1 protein exposed on the surface of monocytes;
(2) the method according to (1), wherein the osteoclasts are cells cultured in vitro;
(3) the in vitro cultured cells of osteoclasts obtainable by contacting monocytes with monoclonal antibody that binds to a portion of FRP-1 protein exposed on the surface of monocytes;
(4) the in vitro cultured cells according to (3), which is derived from human;
(5) an agent for inducing differentiation of monocytes to osteoclasts which comprises monoclonal antibody that binds to a portion of FRP-1 protein exposed on the surface of monocytes as an active ingredient;
(6) a method for screening a ligand that binds to FRP-1 protein, which comprises steps of treating monocytes with a test sample and detecting differentiation of monocytes to osteoclasts;
(7) a ligand that binds to FRP-1 protein and can be isolated by the method according to (6).
(8) the ligand according to (7), which is a naturally-occurring substance.